Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

trigger this global remodeling. The fetal retinal sheet transplants as a natural replacement for lost photoreceptors may provide the most suitable mechanical support to prevent retinal collapse.

7. FUTURE DIRECTIONS

Fetal retinal sheet transplantation has been shown to have a rescue and restoration effect on visual function in various rodent models with retinal degeneration. Clinical studies indicate that fetal retinal transplants with its RPE can survive without apparent rejection and show improvement in visual acuity in some patients. Additional work is in progress attempting to confirm the reproducibility of these results and enhance the magnitude of the effect. Current and future studies are aiming at improving the interconnections between the transplant and the host retina. Elimination of the inner limiting membrane on the surface of the donor retina that can act as a barrier might lead to improvements in the connectivity between transplant and host. Connectivity could be also improved using appropriate factors that promote neurogenesis and reduce glial scars and trauma.

8. ACKNOWLEDGEMENTS

Supported by Foundation Fighting Blindness, Foundation for Retinal Research, Fletcher Jones Foundation, NIH EY03040 and Private Funds, and an NEI travel grant to BT. For space reasons, only some of the people involved in the studies discussed in this chapter can be mentioned. We thank Zhenhai Chen, Xiaoji Xu, Lilibeth Lanceta, and Betty Nunn for technical assistance. Guanting Qiu (Doheny Eye Institute, Los Angeles, CA); Shinichi Arai (Niigata University, Niigata, Japan); Botir T. Sagdullaev (Washington University, St. Louis); Norman D. Radtke, Heywood M. Petry, Maureen A. McCall, Peng Yang (University of Louisville); Gustaw Woch (Hershey Medical School, Hershey, PA); and Sherry L. Ball (Cleveland University, Cleveland, OH) contributed to the work presented in this paper. We thank Matthew M. LaVail, UCSF, for the founder breeding pairs of transgenic S334ter rats, and Eric Sandgren, University of Wisconsin, for founder breeders of transgenic hPAP rats.

Robert Aramant and Magdalene Seiler have a proprietary interest in the implantation instrument and procedure.

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Figure 53.2. The adult human eye contains highly proliferative undifferentiated cells.

sion of expanded and differentiated hRPCs from two different donors (E and L). During expansion (i.e. in the presence of EGF and 10% FBS), RSCs expressed markers present in neural and other stem cells: as evidenced by immunostaining (Figure 53.2E), nestin was detected at a high expression level during the expansion, validating the microarray approach. We also observed the expression of ABCG2 which is present in hematopoietic stem cells (Zhou et al, 2003), or Bmi1 which is required for hematopoietic stem cell (Park et al, 2003, Lessard et al, 2003) and neural stem cell renewal (Molofsky et al, 2003), or nucleostemin which is necessary for NSC renewal (Tsai and McKay, 2002). The expression of Bmi1 was confirmed by RT-PCR (data not shown). Thus, the highly proliferative cells derived from the adult human eye share common characteristics with other stem cells.

The ability of these cells to differentiate into the different cell types composing the organ from which they have been isolated is one of the stem cell characteristics. Thus, to portray the progeny of the RSCs we also analysed their gene expression profile after differentiation (i.e. after withdrawal of FBS and stimulation by EGF). The cells express genes that have different functions in neurons such as genes coding for the skeleton (Map2, Tau), for the synaptic vesicles proteins (SNAPAP). Moreover specific markers of retinal cells are also expressed as shown by the presence of RPE65 (protein are expressed in retinal pigmented cells), opsin, and peripherin (specific to photoreceptors). In one of the cell line, we also detected by immunostaining, cells that have differentiated into retinal neurons and which expressed specific proteins such as calbindin for horizontal cells (Figure 53.3A), syn-

Figure 53.3. The RSCs differentiate into retinal neurons.

taxin for amacrine cells (Figure 53.3B), recoverin for photoreceptor and some bipolar cells (Figure 53.3C) and vimentin for glial cells (data not shown).

4. CONCLUSION

We showed that adult human eye contains cells that can easily be expanded (one cell giving rise to more than 100 billion cells) and share common characteristics with other stem cell populations. Furthermore, after differentiation, those cells expressed markers of several retinal cell types such as pigmented epithelium (RPE65), glia (vimentin) or retinal neurons (opsin, recoverin). The potential of generating retinal neurons in vitro is a promising tool. Indeed the human macula is composed of 200,000 cells which represent only 1/1,000,000 of the total number of cells that we can generate from one RSC. Thus, RSCs could circumvent the problem of the cell number limitation for transplantation studies and could serve to dissect the mechanisms leading to the generation of neurons and their survival.

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Table 54.1. Comparison of embryonic vs. adult stem cell characteristics.

capacity for self-renewal. There are advantages and disadvantages associated with the use of both embryonic (ESC) and adult stem cells (ASC) in cell-based therapeutic strateies to treat degenerative disease and these have been summarised in Table 54.1. It is beyond the scope of this article to comprehensively review these aspects in detail and we therefore refer the reader to several excellent articles and reviews specifically dealing with these topics (Daar et al., 2004; Hochedlinger et al., 2004).

The potential of stem cells in a cell replacement strategy for the treatment of degenerative disease has been recognised from the results of both in vitro and in vivo analyses. In vitro analyses of photoreceptor sheets, retinal explants, primary retinal pigment epithelium (RPE), cultured RPE, and of stem cells themselves have contributed to the belief that stem cells may have a role in the treatment of retinal degenerations. Photoreceptor differentiation has been analyzed by co-culture of stem cells with retinal cells from the embryonic retina (Belliveau et al., 2000), neonatal retina (Ahmad et al., 1999; Ahmad et al., 2000), RPE (Chiou et al., 2005), and stromal cells (Hirano et al., 2003). Other studies inducing the differentiation of marrow stromal cells (MSC) into mesodermal, neuroectodermal and endodermal derivatives have also substantiated the ability of stem cells to differentiate into the lineages required for cell replacement in the degenerate retina (Jiang et al., 2002; Woodbury et al., 2002). Perhaps one of the most informative studies demonstrating the potential of stem cells for the treatment of retinal degenerative disease is that of Zhao et al. (2002) where they showed that ESC-derived neural cells express photoreceptor regulatory genes.

Investigations of the viability, integration, and functionality of transplanted retinal tissue have shown promise for possible treatment of retinal degenerations. Transplanted tissue has included fetal (Sagdullaev et al., 2003), and adult sheets or dissociated photoreceptors (Gouras et al., 1991), RPE (Gouras et al., 1989; Wang et al., 2004), full-thickness neuroretina (Ghosh et al., 2004), and microaggregates of neural retina (Gouras and Tanabe, 2003). In a recent study, the activity in the superior colliculus was analysed following transplant of embryonic rat retinal sheets to the retina of a transgenic rat with photoreceptor degeneration where the transplant was shown to improve visual function (Sagdullaev et al., 2003). Using a mouse model, similar results were found to be associated with the rescue of host cones in the transplant recipients (Arai et al., 2004). Whilst such results are promising, there is evidence that rescue of visual function may only be possible if the RPE cells are transplanted prior to the major loss of photoreceptors (Li and Turner, 1991; Castillo

et al., 1997). The small number of cells harvested from fetal ocular tissue, and the ethical issues surrounding the use of fetal tissue, present major obstacles to the clinical application of such cell replacement therapy.

3. RECENT INVESTIGATIONS USING STEM CELLS TO REPAIR THE DEGENERATE RETINA

3.1. Transplantation of Embryonic Stem Cells

A number of different laboratories have studied the effects of transplanting ESC into the subretinal space (Schraermeyer et al., 2001; Arnhold et al., 2004; Haruta et al., 2004) and into the vitreous of the eye (Hara et al., 2004; Meyer et al., 2004). Transplantation of undifferentiated murine and primate ESC into the dystrophic RCS rat (Schraermeyer et al., 2001; Haruta et al., 2004) both resulted in establishing a multilayered photoreceptor outer nuclear layer (ONL) compared to a non-existent ONL in the sham-injected animals. Haruta and associates (2004) also presented evidence for a concomitant improvement in visual function. Recently, Meyer et al. (2004) transplanted retinoic acid-induced, ESC-derived, neural precursors intravitreally into five week old rd1 mice. A small proportion of the transplanted cells were able to cross the inner limiting membrane, incorporate into the neural retina and to differentiate into cells expressing neural cell markers whilst resembling neural cell morphology. In the age-matched normal C57BL/6J mouse model, the donor cells remained in the vitreous distant from the retinal surface. This suggests that chemo-attractant or migration factors are present in the degenerate retinas that are not present in the normal model. Arnhold et al. (2004) also differentiated the ESC into neural precursors prior to subretinal transplantation into rhodopsin-knockout mice. The transplanted cells did not appear to disrupt the cellular laminae until 8 weeks post-transplant when teratomas were seen to affect the choroid, retina, and vitreous of host eyes. Hara et al. (2004) also saw teratoma formation after transplantation of ESC intravitreally in mice and this appears to be a major obstacle to the application of ESC in cell replacement therapy.

3.2. Transplantation of Adult-Derived Stem Cells

3.2.1. Retinal-Derived Stem Cells

As with ESC, the subretinal transplantation of retinal stem cells (RSC) into rats with disease or injury has shown the survival, integration and differentiation into cells expressing markers of the neural retina, including photoreceptors (Akagi et al., 2003; Chacko et al., 2003; Klassen et al., 2004a). Injection into normal animal models appears to inhibit the incorporation of such cells into the retina (Chacko et al., 2000; Chacko et al., 2003). Intravitreal injection of RSC into immunocompetent mice (Coles et al., 2004) and rats (Seigel et al., 1998) have shown that RSC survive, migrate from the vitreous and integrate into the neural retina. The characteristics of retinal and neural stem cells (NSC), their developmental roles, molecular mechanisms known to control their specification, and transplantation of NSC into the retina are all described in a comprehensive review by Klassen et al. (2004b).